+

WO2006028197A1 - Anticorps anti-a33 - Google Patents

Anticorps anti-a33 Download PDF

Info

Publication number
WO2006028197A1
WO2006028197A1 PCT/JP2005/016576 JP2005016576W WO2006028197A1 WO 2006028197 A1 WO2006028197 A1 WO 2006028197A1 JP 2005016576 W JP2005016576 W JP 2005016576W WO 2006028197 A1 WO2006028197 A1 WO 2006028197A1
Authority
WO
WIPO (PCT)
Prior art keywords
antibody
seq
variable region
acid sequence
ferm
Prior art date
Application number
PCT/JP2005/016576
Other languages
English (en)
Japanese (ja)
Inventor
Shiro Kataoka
Takafumi Tomura
Noriko Otani
Original Assignee
Kirin Beer Kabushiki Kaisha
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Kirin Beer Kabushiki Kaisha filed Critical Kirin Beer Kabushiki Kaisha
Priority to EP05778552A priority Critical patent/EP1801208A4/fr
Priority to CN2005800298522A priority patent/CN101010427B/zh
Priority to US11/629,779 priority patent/US7579187B2/en
Priority to AU2005280975A priority patent/AU2005280975B2/en
Priority to CA2579391A priority patent/CA2579391C/fr
Priority to JP2006535830A priority patent/JP4088655B2/ja
Publication of WO2006028197A1 publication Critical patent/WO2006028197A1/fr
Priority to HK07111099.0A priority patent/HK1105993A1/xx
Priority to US12/053,461 priority patent/US7432359B2/en
Priority to US12/421,431 priority patent/US20090299039A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/734—Complement-dependent cytotoxicity [CDC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • the present invention relates to anti-A33 antibodies that specifically bind to A33 antigen. Furthermore, the present invention relates to a preventive or therapeutic agent for diseases caused by cells expressing A33, particularly a malignant tumor therapeutic agent, comprising an anti-A33 antibody as an active ingredient.
  • Cancer is the leading cause of death in Japan, and the number of patients is increasing year by year, and the development of highly effective and safe drugs and treatment methods is strongly desired. Among them, colorectal cancer accounted for 12.2% of all cancers in the survey in fiscal 1999, and the death rate was 3rd for men and 2nd for women. In the future, colon cancer is expected to overtake stomach cancer with morbidity and mortality.
  • Gastric cancer
  • tumor-specific antigens are useful for the treatment of pathological conditions that exhibit the characteristics of heterologous cells.
  • the antibody binds to a tumor-specific antigen, a protein expressed on the cell surface, and effectively targets such cells.
  • tuximab monoclonal antibodies such as humanized antibodies targeting Her2 / neu have been used as target diseases for malignant tumors, and their therapeutic effects have been observed.
  • An antibody is characterized by a long half-life in blood and high specificity to an antigen, and is particularly useful as an antitumor agent. For example, in the case of an antibody that targets a tumor-specific antigen, it is estimated that the administered antibody accumulates in the tumor.
  • mouse antibodies were used as target animals for the first antibody production.
  • the use of mouse antibodies in vivo is limited for a number of reasons.
  • Mouse antibodies that are recognized as foreign by human hosts elicit a so-called “human anti-mouse antibody” or “HAMA” response (see Schiff et al., Cane. Res. (1985), 45, 879-885). ).
  • HAMA human anti-mouse antibody
  • the Fc portion of mouse antibodies is not effective in stimulating human complement or cytotoxic activity.
  • Chimeric antibodies have been developed as one approach to avoid such problems (see European patent applications 120694 and 125023).
  • a chimeric antibody contains a portion of an antibody from two or more species (such as the variable region of a mouse antibody and the constant region of a human antibody). The advantages of such a chimeric antibody retain the characteristics of a mouse antibody, but have human Fc and can stimulate human complement or cytotoxic activity. However, such chimeric antibodies still elicit a “human anti-chimeric antibody” or “HACA” response (see Bruggemann, et al., J. Exp. Med., 170, 2153-2157, 1989).
  • CDR complementarity determining region
  • a mouse anti-A33 antibody and an arsenic antibody against an antigen that is a class I cell membrane protein called “A33” and one of the Ig superfamily and a tumor-specific antigen has been reported (Patent Document 1 and Non-Patent Document). 1 to 5).
  • This antigen is It is known to be related to colon cancer and gastric cancer (see Patent Document 2, Patent Document 3 and Non-Patent Document 6).
  • Phase I clinical trials have been conducted for colon cancer patients using this humanized A33 antibody (see Non-Patent Documents 4 and 5). In the former report of single antibody administration, partial reaction was observed in 1 out of 11 patients who could receive antibody.
  • the humanized A33 antibody showed a very high tumor response in Phase I clinical trials, but both trials produced human anti-humanized antibodies (ie “HAHA”) with a high probability of more than 50%. It was done. Interestingly, HAHA was not observed in patients with high tumor reactivity.
  • Patent Document 1 US Patent No. 5958412
  • Patent Document 2 US Pat.
  • Patent Document 3 US Patent No. 5160723
  • Non-Patent Document 1 King DJ et a British J. Cancer (1995) 72, 1364-1372
  • Non-Patent Document 2 Welt S, et al., J. Clinical Oncology (1994), 12, 1561-1571
  • Non-Patent Document 3 Welt S. et al., J. Clinical Oncology (1996), 14, 1787-1797
  • Non-patent document 4 Welt S. et al., Clinical Cancer Res. (2003), 9, 1338-1346
  • Non-patent document 5 Welt S. et al., Clinical Cancer Res. (2003), 9, 1347-1353
  • the object of the present invention is to convert tumor cells that can bind to and express A33 to ADCC or CDC. It is intended to provide a preventive or therapeutic agent for various malignant tumors such as solid tumors that are currently difficult to treat by developing antibodies that specifically attack using the immune system of and that does not produce HAHA.
  • antibodies targeting the A33 antigen are considered suitable for application as antitumor agents. Moreover, antibodies that do not produce HAHA may have a higher antitumor effect. Therefore, as a result of earnest research on the production of antibodies against A33, the present inventors succeeded in obtaining monoclonal antibodies that exhibit antitumor effects against cancer cells expressing A33, and further, in the variable region of the monoclonal antibodies. The sequence was specified and the present invention was completed.
  • the present invention in its first aspect, is preferably produced by a monoclonal antibody that binds to A33 produced by a mouse-mouse hybridoma, such as 263A17, 125M10AA 125M165 arm, 125M96ABA, 125N26F6AA, 125Q47BA, 125Q54AAAA or 125R5AAAA.
  • Monoclonal antibodies that are antibodies or functional fragments thereof are provided.
  • the type of monoclonal antibody produced by 263A17, 125M10AA, 125M165DAAA, 125M96ABA, 125 ⁇ 6AA, 125Q47BA, 125Q54AAAA or 125R5AAAA is human immunoglobulin G (IgG).
  • 125M10AA, 125M165DAAA, 125M96ABA, 125N2 & F6AA, 125Q47BA, 125Q54AAAA or 125R5AAAA of the above-mentioned high pridors are listed on 1 August 24, 2004, National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center (Tsukuba Sakai Higashi, Ibaraki, Japan) Autonomous number 1 in the middle of the 6th), the order number FERM BP-10107 (for identification: M10), FERM BP-10106 (for identification: M165), FERM BP-10108 (for identification) Indication for: M96), FERM BP-10109 (Indication for identification: N26), FERM BP-10104 (Indication for identification: Q47), FERM BP-10145 (Indication for identification: Q54) and FERM Deposited as BP-10103 (Indication for identification: R5).
  • the antibody of the present invention is an antibody or a functional fragment thereof having a variable region of an antibody produced by the above-mentioned hyperdrioma.
  • the antibody of the present invention also includes an antibody with a modified subclass, wherein the antibody produced by hybridoma 263A17 has a subclass of human IgG1, Human IgG2, human IgG3 or human IgG4 antibody or functional fragment thereof, antibody produced by hybridoma 125M10AA and subclass of human IgG1, human IgG2, human IgG3 or human IgG4 or functional fragment thereof, Antibody produced by the domain 125M165DAAA and subclass is human IgGl, human IgG2, human IgG3 or human IgG4 or a functional fragment thereof, antibody produced by the hybridoma 125M96ABA and subclass is human IgGl, human IgG2 , An antibody that is human IgG3 or human IgG4 or a functional fragment thereof, or an antibody produced by Hypridoma 125N26F6AA, whose subclass is human IgG1, human IgG2, human IgG3 or human IgG4, or
  • the present invention relates to an antibody that binds to A33 or a functional thereof, comprising the variable region of an antibody produced by Hyperidoma 263A17, 125M10AA, 15M165DAAA, 125M96ABA, 125N26F6AA, 125Q47BA, 125Q54AAAA, or 125R5AAAA Provide a fragment.
  • the antibody of the present invention is an antibody having a variable region of the amino acid sequence shown in SEQ ID NOs: 23 and 25 or a functional fragment thereof. In another embodiment of the present invention, the antibody of the present invention is an antibody having a variable region of the amino acid sequence shown in SEQ ID NOs: 27 and 29 or a functional fragment thereof. In another embodiment of the present invention, the antibody of the present invention is an antibody having a variable region of the amino acid sequence shown in SEQ ID NOs: 31 and 33 or a functional fragment thereof. In another embodiment of the present invention, the antibody of the present invention is an antibody having a variable region of the amino acid sequence shown in SEQ ID NOs: 35 and 37 or a functional fragment thereof.
  • the present invention further provides, in another aspect, the above-described antibody or a functional fragment thereof, wherein the antibody suppresses the growth of a tumor (eg, derived from a colon cancer cell line C0L0205 cell transplanted into a nude mouse) or Provide functional fragments thereof.
  • a tumor eg, derived from a colon cancer cell line C0L0205 cell transplanted into a nude mouse
  • the amount of the antibody of the present invention or a functional fragment thereof administered to a test animal carrying a tumor for example, a tumor-bearing experimental animal such as a colon cancer cell-bearing mouse model having a body weight of 20 g
  • the dose is 100 g / body or 5 mg / kg, preferably 10 g / body or 0.5 mg / kg.
  • the antibody of the present invention has any of the following characteristics.
  • ADCC antibody-dependent cellular cytotoxicity
  • CDC complement-dependent cytotoxicity
  • the present invention further provides, in another embodiment, Hypridoma 125M10AA (Accession Number FE ⁇ BP-10107), 125M165DAAA (Accession Number BP BP-10106), 125M96ABA (Accession Number FERM BP-10108), 125N26F6AA (Accession Number FERM BP ⁇ 10109), 125Q47BA (Accession No.FERM BP-10104), 125Q54AAAA (Accession No.FERM BP-10105) and Hypridoma 125R5AAAA (Accession No.FERM BP-10103)
  • a nucleic acid encoding the functional fragment of the antibody or the antibody, a protein encoded by the nucleic acid, an expression vector having the nucleic acid, Escherichia coli, yeast cells, insect cells, mammalian cells having the expression vector And a host selected from the group consisting of plant cells and mammals.
  • the present invention further provides, in another embodiment, a gene encoding an anti-A33 monoclonal antibody from a hyperprideoma selected from the group consisting of hyperprideoma 263M7, 125M10AA, 125M165DAAA, 125M96ABA, 125N26F6AA, 125Q47BA> 125Q54AAAA and hyperprideoma 125R5AAAA
  • a gene encoding a variable region of a heavy chain amino acid sequence and a gene encoding a variable region of a light chain amino acid sequence are isolated, an expression vector having the gene is constructed, and the expression vector is introduced into a host.
  • the host is cultured, the monoclonal antibody is expressed, and the anti-A33 monoclonal antibody or a functional fragment thereof is collected from the resulting host, the culture supernatant of the host, or a culture of the host secretion.
  • the present invention further provides a preventive, therapeutic or diagnostic agent for tumors containing the antibody or functional fragment thereof as an active ingredient.
  • colon cancer colon cancer
  • rectal cancer stomach cancer, knee cancer, breast cancer, melanoma
  • renal cell cancer cervical cancer
  • endometrial cancer ovarian cancer
  • esophageal cancer prostate cancer
  • the present invention further relates to Hypridoma M96 (accession number: FE BP), an antibody that binds to A33, which recognizes the same epitope as that recognized by the antibody produced by Hypridoma M10 (Accession number: FE BP-10107).
  • FIG. 1A shows ADCC activity when each monoclonal purified antibody is used to target COL0205 cells.
  • FIG. 1B shows CDC activity when each monoclonal purified antibody is used to target C0L0205 cells.
  • FIG. 1C shows ADCC activity when each monoclonal purified antibody is used to target NCI-H508 cells.
  • FIG. 1D shows CDC activity when each monoclonal purified antibody is used to target NCI-H508 cells.
  • FIG. 2A shows ADCC activity when a recombinant antibody is used to target C0L0205 cells.
  • FIG. 2B shows CDC activity when a recombinant antibody is used to target COL0205 cells.
  • FIG. 2C shows ADCC activity when recombinant antibodies are used to target NCI-H508 cells.
  • FIG. 2D shows the CDC activity when recombinant antibodies are used to target NCI-H508 cells.
  • Fig. 3A is a photograph showing the results of Western plot analysis using purified and recombinant antibodies.
  • Fig. 3B is a photograph showing the results of Western plot analysis using purified and recombinant antibodies.
  • FIG. 4 is a photograph showing the results of immunohistochemical staining of human colon cancer tissue with purified antibody and recombinant antibody.
  • FIG. 5 is a photograph showing the results of immunohistochemical staining of human normal small intestine tissue with purified antibody and recombinant antibody.
  • FIG. 6 is a photograph showing the results of immunohistochemical staining of human normal colon tissue with purified antibody and recombinant antibody.
  • FIG. 7A is a graph showing the antitumor effect of recombinant antibodies cA33 and rec263 against a mouse tumor-bearing model when COL0205 cells are transplanted.
  • FIG. 7B shows the antitumor effect of recombinant antibodies cA33 and rec263 against a mouse tumor-bearing model when NCI-H508 cells are transplanted.
  • FIG. 7C is a diagram showing the antitumor effect of purified purified hybridoma antibodies 125M10AA, 125M165DAAA and 125M96ABA against a mouse tumor-bearing model when COL0205 cells are transplanted.
  • FIG. 7D is a graph showing the antitumor effect of recombinant antibodies recN26 and recM165 on mouse tumor-bearing models when NCI-H508 cells are transplanted.
  • FIG. 7E is a graph showing the antitumor effect of recombinant antibodies recN26 and recM165 against mouse tumor-bearing models when NCI-H508 cells were transplanted with Matriziel.
  • FIG. 7F is a graph showing the antitumor effect of recombinant antibodies recMlO and recQ54 against mouse tumor-bearing models when NCI-H508 cells were transplanted with Matriziel.
  • mouse anti-A33 antibody and humanized anti-A33 antibody have already been obtained, and mouse anti-A33 antibody (Wel t S. et al., J. Clini cal Oncology (1994), 12, 1561-1571 We lt S. et al., J. Cl onical Oncology (1996), 14, 1787-1797) or humanized A33 antibody (Wel t S. et al., Cl inical Cancer Res. (2003), 9, 1338-1346; Wel t S. et al., Clinical Cancer Res. (2003), 9, 1347-1353), reported that Phase I clinical trials were conducted in patients with colon cancer Has been.
  • HAMA or HAHA is produced in antibody-treated patients with a very high probability, and no subsequent clinical trials have been completed.
  • patients with tumor reactivity in clinical trials of humanized anti-A33 antibody failed to produce HAHA.
  • the novel rabbit anti-A33 monoclonal antibody of the present invention is a complete rabbit antibody, and the antigenicity against a portion consisting of a mouse sequence, which is always a problem with mouse antibodies or humanized antibodies, has already been avoided. That is, in the above-mentioned clinical trial reports, HAHA was produced because a humanized antibody was used. However, since the novel human anti-A33 monoclonal antibody of the present invention is a fully human antibody, the antigenicity of the antibody is avoided, and HAHA is Since it is not produced, a high antitumor effect can be expected for colon cancer patients.
  • Immunoglobulin G (IgG), A (IgA), E (IgE), and M (IgM) are used as the antibody class, and IgG is preferred.
  • IgGK IgG2, IgG3, and IgG4 are used as the IgG subclass, preferably IgGl, IgG2, and IgG4, and more preferably IgGl.
  • the “antibody that binds to A33” in the present invention is reactive to A33 or a part thereof. Or an antibody that recognizes A33 or a part thereof.
  • “Functional fragment” means a part (partial fragment) of an antibody that retains at least one action of an antibody on an antigen. Specifically, F (ab ′) 2 , Fab ′ , Fab, Fv, disulfide bond Fv, single chain Fv (scFv), and polymers thereof [DJ King., Applicat ions and Engineering of Monoclonal Ant ibod ies., 1998 TJ Internat ional Ltd ].
  • a “functional fragment” is a fragment of an antibody that can bind to an antigen.
  • baboon antibody means an antibody that is an expression product of a human-derived antibody gene.
  • a human antibody can be obtained by introducing a human antibody locus as described below and administering an antigen to a transgenic animal having the ability to produce a human-derived antibody. Examples of the transgenic animal include mice.
  • a method for producing a mouse capable of producing a human antibody is described in, for example, International Publication No. W002 / 43478.
  • Examples of the antibody of the present invention include various antibodies that exhibit an antitumor effect at a low concentration against cancer cells expressing A33, which are described in Examples below.
  • the antibody of the present invention includes a heavy chain having an amino acid sequence in which one or several amino acids are deleted, substituted, or added in each amino acid sequence of a heavy chain and / or a light chain constituting the antibody. Alternatively, a monoclonal antibody consisting of a light chain is also included.
  • the partial modification (deletion, substitution, insertion, addition) of the amino acid as described above is performed by partially modifying the base sequence encoding the amino acid sequence. Can be introduced.
  • This partial modification of the nucleotide sequence can be introduced by a conventional method using a known site-specific mutagenesis method [Proc Natl Acad Sci USA., 1984 Vol 81 : 5662].
  • the antibody means that all regions including the heavy chain variable region and the heavy chain constant region constituting the immunoglobulin and the light chain variable region and the light chain constant region are derived from the gene encoding the immunoglobulin. It is.
  • the antibodies of the present invention include antibodies having any immunoglopurin class and isotype.
  • the anti-A33 antibody of the present invention can be produced by the following production method. That is, for example, A33, a part thereof or a part thereof and an appropriate agent for enhancing the antigenicity of the antigen Conjugates with carrier substances (eg bovine serum albumin), together with immunostimulants (eg, Freund's complete or incomplete adjuvant), if necessary, for non-human mammals such as baboon antibody producing transgenic mice Immunize.
  • A33 can be either natural A33 or recombinant A33.
  • immunization can be carried out by introducing a gene encoding A33 and administering animal cells overexpressing A33 on the cell surface.
  • Monoclonal antibodies are obtained by culturing a hybridoma obtained by fusing an antibody-producing cell obtained from an immunized animal with a myeloma cell (myeloma cell) that does not have autoantibody-producing ability, and used as an antigen for immunization. It can be obtained by selecting a clone that produces a monoclonal antibody exhibiting a specific affinity for it.
  • the antibodies of the present invention may be modified by genetic engineering modifications well known to those skilled in the art (eg, European patents).
  • ADCC is induced by binding to the antibody constant region via Fc Receptor expressed on the surface of Macrophage, NK cells, neutrophils, etc., and activation of the recognized cell.
  • CDC refers to the cytotoxic activity caused by the activated complement system when an antibody binds to an antigen.
  • an anti-cancer drug if the antibody alone has no cell death-inducing activity, it depends on antibody-dependent cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) via Fc receptors.
  • ADCC antibody-dependent cytotoxicity
  • CDC complement-dependent cytotoxicity
  • An antibody having antitumor activity is desirable, but if the antibody alone has cell death-inducing activity, an antibody having a low degree of binding to the Fc receptor may be more desirable.
  • antibodies that do not have ADCC activity or CDC activity are desirable, such as when sterically inhibiting only the binding between T cells and antigen-presenting cells.
  • ADCC activity or CDC activity can cause toxicity
  • an antibody that avoids the activity causing toxicity by changing the Fc portion mutation or subclass may be desirable.
  • Myeloma cells are prepared (4) ) Cell fusion between antibody-producing cells and myeloma, (5) Selection of the hyperidoma group producing the desired antibody, (6) Division into single cell clones (cloning), (7) In some cases, Culture of high-pridoma to produce a large amount of monoclonal antibodies, or breeding of animals transplanted with high-pridoma. (8) Examination of physiological activity and recognition specificity of monoclonal antibodies produced in this way. Is a sign test Tests for drug properties, etc.
  • A33 has a polymorphism
  • the antibody of the present invention recognizes and binds to all the currently known A33 polymorphisms, so that the present invention can be used regardless of differences in the A33 type of patients.
  • Therapeutic / preventive agents containing these antibodies work effectively.
  • a method for producing an anti-A33 monoclonal antibody will be described in detail according to the above steps.
  • the method for producing the antibody is not limited thereto, and for example, antibody-producing cells other than spleen cells and myeloid cells may be used. it can.
  • a transformant itself obtained by incorporating DNA encoding A33 into an expression vector for animal cells, introducing the expression vector into animal cells, and the like can be used.
  • the primary structure of the A33 protein is known [GenBank access ion No. NPJ05305, SEQ ID NO: 12]
  • a peptide is chemically synthesized from the amino acid sequence of A33 by a method well known to those skilled in the art, It can also be used as 'As an immunogen, cells in which the entire length of A33 is introduced into FM3A cells or L929 cells and A33 is excessively expressed on the cell surface are also effective.
  • p A EGFP-N1-A33 is a DNA encoding the A33 protein, and is an animal cell expression vector p A EGFP-N1 (modified pEGFP-N 1 [Becken. Dickinson-Biosciences, Clontech] In which the region encoding EGFP protein is deleted).
  • DNA encoding A33, vector, host, etc. are not limited to these.
  • a transformed strain obtained by transforming FM3 A cells or L929 cells with P ⁇ EGFP-N 1 -A33 is cultured, and neo-obtained in cells into which the pAEGFP-N1 vector has been inserted. It is possible to produce FM3A cells or L929 cells that overexpress A33 on the cell surface using the characteristics of mycin resistance and confirmation of A33 expression using mouse A33 antibody (AKC No. HB-8779) as an index. it can.
  • the antigen obtained in (1) is mixed with an adjuvant such as Freund's complete or incomplete adjuvant, or karimiyoban, and an experimental animal is immunized as an immunogen.
  • an adjuvant such as Freund's complete or incomplete adjuvant, or karimiyoban
  • Transgenic mice having the ability to produce human-derived antibodies are most preferably used as experimental animals. Such mice are described in Totsuka et al. [Tomizuka. Et al., Proc Natl Acad Sc i USA., 2000 Vol 97: 722].
  • the immunogen administration method for mouse immunization may be subcutaneous injection, intraperitoneal injection, intravenous injection, intradermal injection, intramuscular injection, footpad injection, but intraperitoneal injection, footpad injection or intravenous injection Is preferred. Immunization can be performed once or multiple times at appropriate intervals (preferably at intervals of 2 to 4 weeks). Thereafter, the antibody titer against the antigen in the serum of the immunized animal is measured, and if an animal with a sufficiently high antibody titer is used as a source of antibody-producing cells, the effect of the subsequent operation can be enhanced. In general, antibody-derived cells derived from animals 3 to 5 days after the final immunization are preferably used for subsequent cell fusion.
  • the antibody titers used here include radioisotope immunoassay (hereinafter referred to as “RIA method”), solid-phase enzyme immunoassay (hereinafter referred to as “ELISA method”), fluorescent antibody method, passive blood cell Various known techniques such as the agglutination reaction method can be mentioned, but the RIA method or the ELISA method is more preferable from the viewpoint of detection sensitivity, rapidity, accuracy, and possibility of automation of operation.
  • RIA method radioisotope immunoassay
  • ELISA method solid-phase enzyme immunoassay
  • fluorescent antibody method passive blood cell
  • passive blood cell Various known techniques such as the agglutination reaction method can be mentioned, but the RIA method or the ELISA method is more preferable from the viewpoint of detection sensitivity, rapidity, accuracy, and possibility of automation of operation.
  • Measurement of the antibody titer in the present invention can be carried out according to the procedure described below, for example, by ELISA.
  • a solid phase surface such as a 96-well plate for ELISA, and the solid surface on which no antigen is adsorbed is covered with a protein unrelated to the antigen, such as ushi serum albumin (BSA).
  • BSA ushi serum albumin
  • the sample is contacted with a serially diluted sample (eg, mouse serum) as a primary antibody, and the anti-A33 antibody in the sample is bound to the antigen.
  • an antibody against a human antibody labeled with an enzyme as a secondary antibody is added and bound to the human antibody.
  • the substrate of the enzyme is added, and the change in absorbance due to color development based on the decomposition of the substrate is measured. Is calculated.
  • mice having no autoantibody-producing ability derived from mammals such as mice, rats, guinea pigs, hamsters, rabbits or humans can be used.
  • cell lines obtained from mice for example, 8-Azaguanine resistant mice
  • DMEM Dulbecco's Modified medium
  • normal medium eg, DMEM medium containing 10% FCS
  • Antibody-producing cells are plasma cells and lymphocytes that are precursor cells thereof, which may be obtained from any part of the individual, and generally include the spleen, lymph nodes, bone marrow, tonsils, terminal blood, or these Spleen cells are most commonly used, although they can be obtained from appropriate combinations.
  • Spleen cells and myeloma are thoroughly washed with serum-free medium (eg, DMEM) or phosphate buffered saline (hereinafter referred to as “PBS”), and the ratio of the number of spleen cells to myeloma is 5: 1
  • serum-free medium eg, DMEM
  • PBS phosphate buffered saline
  • IL-6 normal medium
  • HAT hypoxanthine, aminopterin and thymidine
  • IL-6 chick interleukin-6
  • the myeloma cell is an 8-azaguanine resistant strain, that is, when it is a hypoxanthine'guanine-phospholiposyltransferase (HGPRT) deficient strain
  • HGPRT hypoxanthine'guanine-phospholiposyltransferase
  • the myeloma cell that has not fused, and the fusion cell between myeloma cells Cannot survive in medium containing HAT.
  • fused cells between antibody-producing cells, or a hyperidoma between antibody-producing cells and myeloma cells can survive, but fused cells between antibody-producing cells have a lifetime. Therefore, by continuing the culture in the HAT-containing medium, only the hyperidoma, which is a fusion cell of the antibody-producing cell and the myeloma cell, survives, and as a result, the hyperidoma can be selected.
  • Hypridoma grown in colonies change the medium to a medium in which aminopterin is removed from the HAT medium (hereinafter referred to as “HT medium”). Thereafter, a part of the culture supernatant is collected, and the anti-A33 antibody titer is measured by, for example, ELISA.
  • HT medium a medium in which aminopterin is removed from the HAT medium
  • ELISA anti-A33 antibody titer
  • the method using the 8-azaguanine-resistant cell line has been exemplified above, but other cell lines can be used depending on the selection method of the hyperidoma, and in this case, the composition of the medium used also changes.
  • This cloning method includes limiting dilution, in which one well of the plate is diluted so that one hyperidoma is contained, the soft agar method in which the colonies are collected by culturing in a soft agar medium, and a micromanipulator. Examples include the method of taking out one cell at a time and culturing it, and the “So-Ichiyu clone” in which one cell is separated by Cell Soryu, but the limiting dilution method is simple and often used.
  • cloning by limiting dilution Repeat 2 to 4 times and select the one with stable antibody titer as the anti-A33 monoclonal antibody-producing hybridoma strain.
  • mouse anti-A33 monoclonal antibody of the present invention mouse-mouse hyperpridoma 125M10AA ⁇ 125M165DAAA, 125M96ABA ⁇ 125N26F6AA, 125Q47BA, 125Q54AAAA, or 125R5AAAA, is an incorporated administrative agency industry on August 24, 2004 The Research Center for Biological Biology (in Japan, 1st, 1st, 1st, 1st, 1st, Tsukuba City, Ibaraki Prefecture, Japan), with the accession numbers FERM BP-10107 (indication for identification: M10), FERM BP- 10106 (Indication for identification: M165), FE BP-10108 (Indication for identification: M96), FERM BP-10109 (Indication for identification: N26), FERM BP-10104 (Indication for identification : Q47), FERM BP-10105 (Indication for identification: Q54) and FERM BP-10103 (Indication for identification: R5).
  • the hybridoma is cultured by changing the medium from HT medium to normal medium.
  • Large-scale culture is performed by rotary culture using a large culture bottle, spinner culture, or culture using a holo-fiber system.
  • An anti-A33 monoclonal antibody can be obtained by purifying the supernatant in this large-scale culture using a method well known to those skilled in the art, such as gel filtration.
  • ascites containing a large amount of anti-A33 monoclonal antibody can be obtained by growing the hybridoma in the abdominal cavity of the same strain (e.g. BALB / c) or nu / nu mouse, rat, guinea pig, hamster or rabbit.
  • a commercially available monoclonal antibody purification kit for example, AbTrap GI I kit; manufactured by Amersham Pharmacia Piotech
  • the like can be used as a simple purification method.
  • the monoclonal antibody thus obtained has a high antigen specificity for A33.
  • the isotype and subclass of the monoclonal antibody thus obtained can be determined as follows. 'First, the identification method
  • the octelloni method is simple, but concentration is necessary when the concentration of the monoclonal antibody is low.
  • the ELISA method or the RIA method is used, the culture supernatant is reacted with the antigen-adsorbing solid phase as it is, and further, antibodies corresponding to various immunoglobulin isotypes and subclasses are used as secondary antibodies. It is possible to identify isotypes and subclasses.
  • protein quantification can be carried out by a foreign lawy method, a method of calculating from the absorbance at 280 nm [1.4 (OD280) nimunoglopurin lmg / mL], and the like.
  • Identification of a monoclonal antibody recognition epitope can be performed as follows. First, various partial structures of molecules recognized by monoclonal antibodies are prepared. In preparing the partial structure, a method for preparing various partial peptides of the molecule using a known oligopeptide synthesis technique, and a DNA sequence encoding the target partial peptide using a gene recombination technique are suitable expression plasmids. However, it is common to use both in combination for the above purpose. For example, after preparing a series of polypeptides sequentially shortened by an appropriate length from the C-terminus or N-terminus of an antigen protein using genetic recombination techniques well known to those skilled in the art, the reactivity of monoclonal antibodies against them is examined. Determine the rough recognition site.
  • kits for example, SPOTs kit (manufactured by Dienosis Biotechnologies), a series of multipin peptide synthesis kits using multipin synthesis method (manufactured by Chiron)) Etc.
  • SPOTs kit manufactured by Dienosis Biotechnologies
  • a series of multipin peptide synthesis kits using multipin synthesis method manufactured by Chiron
  • a gene encoding rabbit monoclonal antibody is cloned from antibody-producing cells such as hypridoma and incorporated into an appropriate vector, which is then used as a host (eg, mammalian cell line, E. coli, yeast cell, insect cell, plant cell). Etc.) and the residue Recombinant antibodies produced using gene recombination technology can also be prepared
  • the present invention relates to a nucleic acid comprising an antibody gene sequence possessed by a hyperprideoma that produces the antibody of the present invention, particularly a heavy chain variable region and a light chain variable region of an antibody produced by the below-described hyperpride monomer of the present invention.
  • nucleic acids are also included.
  • the nucleic acid includes DNA and RNA.
  • DNAs encoding the L chain V region, L chain C region, H chain V region and H chain C region of the monoclonal antibody are prepared by PCR or the like. Is adopted.
  • an oligo DNA designed from an anti-A33 antibody gene or an amino acid sequence can be used, and a DNA prepared from a hybridoma can be used as a cage. These DNAs are incorporated into one appropriate vector and introduced into a host for expression, or each of these DNAs is incorporated into an appropriate vector for co-expression.
  • a phage or a plasmid capable of autonomously growing in a host microorganism is used.
  • plasmids include plasmids derived from E. coli, Bacillus subtilis, or yeast, and examples of phage MA include ⁇ phage.
  • the host used for transformation is not particularly limited as long as it can express the target gene. Examples include bacteria (E. coli, Bacillus subtilis, etc.), yeast, animal cells (COS cells, CH0 cells, etc.), and insect cells.
  • bacteria E. coli, Bacillus subtilis, etc.
  • yeast yeast
  • animal cells COS cells, CH0 cells, etc.
  • insect cells insect cells.
  • Methods for introducing a gene into a host are well known, and any method (for example, a method using calcium ion, an electoporet method, a spheroplast method, a lithium acetate method, a calcium phosphate method, a lipofuxion method, etc.) can be mentioned.
  • Examples of the method for introducing a gene into an animal described later include a microinjection method, a method for introducing a gene into ES cells using a lipopoxion method, and a nuclear transfer method.
  • the antibody is collected by disrupting the cells or cells.
  • the target antibody is produced outside the cells or cells, use the culture medium as it is, or remove the cells or cells by centrifugation or the like. Thereafter, the target antibody is isolated and purified from the culture by using a general biochemical method using various chromatographs used for protein isolation and purification alone or in appropriate combination. Can do.
  • the animal host in which the gene of the target antibody has been incorporated into the endogenous gene such as Transgeneic Cushion, Transgeneic Goat, ⁇ Lanceienic Hedge or Transgenic Beverage. It is also possible to obtain a large amount of monoclonal antibodies derived from the antibody gene from milk produced and secreted from the transgenic animal (Wright, G., et al.
  • Hypridoma When cultivating Hypridoma in vitro, the hybridoma is grown, maintained and stored according to various conditions such as the characteristics of the cell type to be cultured, the purpose of the research and the culture method, etc. It can be carried out using a known nutrient medium such as that used to produce null antibodies or any nutrient medium prepared and prepared from a known basic medium.
  • the antibody of the present invention has any of the following characteristics.
  • ADCC antibody-dependent cellular cytotoxicity
  • CDC complement-dependent cytotoxicity
  • antibodies examples include antibodies produced by the hybridomas 263A17, 125M10AA, 125M165 ⁇ , 125M96ABA, 125N26F6AA, 125Q47BA, 125Q54AAAA, and hyperidoma 125R5AAAA, and 125M10AA, 125M165DAAA. As of August 24, 2004, 5AAAA will be assigned to the Patent Organism Depositary Center of the National Institute of Advanced Industrial Science and Technology (1st East, 1st Street, Tsukuba City, Ibaraki, Japan, 1st 6th).
  • Formulations containing the human anti-A33 antibody of the present invention are also included within the scope of the present invention.
  • Such formulations preferably contain a physiologically acceptable diluent or carrier in addition to the antibody, and may be a mixture with other antibodies or other agents such as antibiotics.
  • Suitable carriers include, but are not limited to, physiological saline, phosphate buffered saline, phosphate buffered saline glucose solution, and buffered saline.
  • the antibody may be freeze-dried (freeze-dried) and reconstituted by adding a buffered aqueous solution as described above when needed.
  • Such prophylactic or therapeutic agents can be administered in various forms, such as tablets, capsules, granules, powders, syrups, etc. Oral administration or parenteral administration such as injection, infusion, suppository and the like can be mentioned.
  • oral administration is about 0.01 mg to 1000 ing per day for adults, which are administered once or divided into several times be able to.
  • parenteral administration about 0.01 mg to 1000 mg can be administered once by subcutaneous injection, intramuscular injection or intravenous injection.
  • the present invention also includes a method for preventing or treating the above-mentioned diseases using the antibody or pharmaceutical composition of the present invention, and the present invention further includes the use of the antibody of the present invention for the manufacture of a preventive or therapeutic agent for the above-mentioned diseases. Include.
  • Tumors that can be prevented or treated with the antibodies of the present invention or functional fragments thereof are colon cancer, colon cancer, rectal cancer, stomach cancer, knee cancer, breast cancer, melanoma, renal cell cancer, cervical cancer, endometrium Cancer, ovarian cancer, esophageal cancer, prostate cancer, testicular cancer, mesothelioma cancer, etc.
  • the tumor when applying the antibody of the present invention is not limited to one type, and may be a combination of multiple types of tumors. 3. Formulation example
  • the molecules of the present invention are provided for use as ampoules in sterile solutions or suspensions dissolved in water or other pharmacologically acceptable solutions.
  • a sterile powder preparation (preferably lyophilized the molecule of the present invention) may be filled in an ampoule and diluted with a pharmaceutically acceptable solution at the time of use.
  • Type Culture Collection (ATCC) No. HB-8779) was purchased and hypridoma was cultured according to the ATCC instruction manual. This hyperidoma was stored frozen. afterwards,
  • AS33 Hypridoma was conditioned to 10% Low IgG Fetal Bovine Serum (High Clone) -containing eRDF medium (Kyokuto Pharmaceutical). This acclimated high-pridoma is kept frozen. Existed.
  • Ushiinsulin (5 g / ml, manufactured by Gibco Beer Luel)
  • human transferrin (5 ⁇ g / ml, manufactured by Gibco PRL)
  • ethanol Miner (0.01 Sigma
  • sodium selenite 2.5xl (T 5 mM, Sigma)
  • 13 ⁇ 4 Low IgG Fetal Bovine Serum (Haikuguchi)
  • the culture supernatant was recovered and the concentration of the purified hybridoma-derived antibody in the recovered supernatant was measured by measuring the absorbance at 280 nm and calculating 1 mg / mL as 1.40 D.
  • Example 2 Preparation of chimeric anti-A33 antibody
  • a chimeric anti-A33 antibody in which the heavy chain and light chain constant regions were human IgG1 was prepared.
  • DMEM medium Gibco®
  • the total RNA was purified according to the protocol using the RNA extraction reagent IS0GEN (Nitsubon Gene), then 01 igotexTM-dT30 ⁇ Super> (Takara Bio)
  • the polyA + RNA was purified using the SMARi RACE cDNA Amplification Kit (Becton / Dickinson / Bioscience 'Clontech) using the resulting polyA + RNA (2.5MS) as a material. Cloning experiments were performed to obtain the variable region cDNA
  • reaction solution having the above composition was incubated at 70 ° C for 2 minutes, the following reagents and enzymes were added and incubated at 42 ° C for 1.5 hours to synthesize cDNA.
  • mouse anti-A33 antibody heavy chain hereinafter also referred to as heavy chain variable region and light chain (hereinafter, light chain also referred to as L chain) variable region DNA 3 ' PCR primer specific to each end (for H chain: GPAHvR3Nlie (5 '-GCC CTT GGT GCT AGC TGAAGAGAC GGT GAC CAG AGT CCC TTG-3') (SEQ ID NO: 1)), for L chain: GPALvR3Bsi, ( 5'-GTG CAC GCC GCT GGT CAG GGC GCC TG-3 ') (SEQ ID NO: 2)) and the UPM primer included in the SMART RACE cDNA Amplification Kit (prepared at the 5' end of the synthesized cDNA) Oligonucleotide complementary to the common sequence) is used as a primer set to amplify the H chain leader sequence and variable region (hereinafter also referred to as HV) and L chain leader sequence and variable
  • GSP Gene specific primers
  • the amplification reaction conditions for thermal cycling were as follows.
  • the amplified PCR fragment was recovered by ethanol precipitation, recovered by agarose gel electrophoresis, and purified by QIAduick Gel Extraction Kit (Qiagen), which is a DNA purification kit using a membrane.
  • Qiagen is a DNA purification kit using a membrane.
  • the purified HV and LV amplified fragments were subcloned into the PCR Blunt-T0P0 vector of Zero Blunt TOPO PCR Cloning Kit (manufactured by Invitrogen), respectively.
  • the plasmid DNA of the resulting clone was inserted into the base of insert MA. The sequence was analyzed.
  • M13FW (5′-GTAAAACGACGGCCAGTG-3 ′) (SEQ ID NO: 3) and M13RV (5′-CAG GAA ACA GCT ATG AC-3 ′) (SEQ ID NO: 4) were used as primers for determining the DNA base sequence.
  • the amino acid sequence of the antibody encoded in the determined HV and LV gene regions is the mouse anti-A33 antibody reported by KingD. J. et al. (Br J Cancer. 1995 Dec; 72 (6): 1364-72) Completely matched the amino acid sequence of the variable region.
  • CTTAGTT-3 ' (SEQ ID NO: 5) and primer GMHvR3Nhe (SEQ ID NO: 1)) were used to amplify mouse anti-A33 antibody HV by PCR (94 ° C 3 minutes ⁇ 94 ° C 10 seconds, 68 ⁇ 45 seconds)
  • the amplified fragment of HV was purified and then subcloned with PCI Blim ⁇ T0P0 vector.
  • the DNA base sequence analysis of the inserted portion of the subclone was performed, and a plasmid DNA having a sequence as designed that was not different from the caged gene sequence was selected.
  • the plasmid DNA is converted into restriction enzymes Sail and Nliel And digested with agarose gel electrophoresis to recover and purify about 440 bp of DNA.
  • the vector N5KG ⁇ Val Lark modified vector of I DEC Pharmaceuticals, N5KG1 (US patent 6001358) was similarly treated with restriction enzymes Sail and Nhel, and then dephosphorylated as Alkal.
  • restriction enzymes Sail and Nhel After treatment with ine Phosphatase (E. coli C75) (manufactured by Carabio Co., Ltd.), about 8.9 kb of DNA was recovered by agarose gel electrophoresis and a DNA purification kit. These two fragments were subjected to a ligation reaction with DNA Ligation Kit Ver. 2.1 (manufactured by Yubara Bio Inc.) and then introduced into E. coli DH5 Q! To obtain a transformant.
  • N5KGl_GPA33Hv (clone # 2), a clone into which the target HV was inserted, was selected by screening transformants.
  • this plasmid DNA was sequentially digested with restriction enzymes Bgl II and BsiWI, further dephosphorylated, and then subjected to an approximately 9.2 kb vector.
  • One DNA was purified.
  • plasmid DNA containing LV of mouse anti-A33 antibody was used as a saddle, and the LV region was amplified by PCR.
  • GPALv2FBgl (5′-AGA GAG AGA GAT CTC TCA CCA TGG GCA TCA AGA TGG AGT TTC AG-3,) (SEQ ID NO: 6) and GPALvR3Bsi (SEQ ID NO: 2) were used as a primer set for amplification.
  • the purified LV amplified fragment was subcloned with PCR 4 Blunt-T0P0.
  • DNA sequence analysis of the inserted portion was performed, and plasmid ⁇ A having a sequence as designed that was not different from the ⁇ type gene sequence was selected.
  • the DNA was digested with restriction enzymes Bglll and BsiWI, and about 400 b of DNA was recovered and purified by agarose gel electrophoresis.
  • This DM was ligated to the above-mentioned N5KG and A33Hv vector fragment digested with restriction enzymes Bglll and BsiWI, and introduced into E. coli to obtain a transformant.
  • the transformant was screened and a clone into which the target LV was inserted, N5KG and GPA33HvLv (clone # 2) were selected.
  • the finally obtained chimeric anti-A33 antibody expression plasmid DNA was purified in large quantities, and it was confirmed that there were no mutations in the L- and H-chain DNA fragment insertion fragments and the DNA base sequence around the insertion site.
  • the DNA encoding the heavy chain variable region and the light chain variable region of the chimeric anti-A33, and the amino acid sequences of the heavy chain variable region and the light chain variable region are shown below, respectively.
  • VPFAYWGQGT LVTYSSAS VPFAYWGQGT LVTYSSAS.
  • CAACAGAAAC CAGGGCAGTC TCCTAAAACA CTGATTTACT TGGCCTCCAA CCGGCACACT
  • the boundary between the antibody variable region and the antibody constant region in the heavy chain nucleic acid sequence is located between the 408th adenine (A) and the 409th guanine (G), and the heavy chain amino acid sequence (SEQ ID NO: 8).
  • the boundary between the signal sequence and the antibody variable region in the heavy chain nucleic acid sequence is located between the 57th thymine (T) and 58th guanine (G), and the heavy chain amino acid sequence (SEQ ID NO: 8 ) Is located between the 19th cysteine (C) and the 20th glutamic acid (E).
  • the nucleic acid sequence (SEQ ID NO: 7) of the variable region of the chimeric anti-A33 antibody heavy chain is from the 58th guanine (G) to the 408th adenine (A).
  • the amino acid sequence of the variable region of the heavy chain (SEQ ID NO: 8) is from the 20th glutamic acid (E) to the 6th serine (S).
  • the nucleic acid sequence (SEQ ID NO: 9) of the variable region of the light chain of the chimeric anti-A33 antibody is from the 73rd guanine (G) to the 393rd adenine (A).
  • the amino acid sequence of the light chain variable region (SEQ ID NO: 10) is from the 25th aspartic acid (D) to the 131st lysine (K).
  • Table 5 below shows the base sequence of the synthetic DNA.
  • the chimeric anti-A33 recombinant antibody expression vector thus prepared was introduced into a host cell to produce a recombinant antibody-expressing cell.
  • Host cells for expression include, for example, dhfr-deficient strains of CH0 cells (ATCC CRL-9096), CHO-Ras (Katakura Y., et al., Cytotecnology, 31: 103-109, 1999), HEK293T ( ATCC CRL-11268) is used.
  • the vector was introduced into the host cell by electroporation or lipofection.
  • electroporation approximately 2 g of the antibody expression vector was linearized with a restriction enzyme, and the gene was introduced into 4 ⁇ 10 6 CH0 cells under conditions of 350 V and 500 F using a Bio-Rad electroporator. l Seeded on a culture plate. Lipofection was performed according to the manual using LipofectAMINE Plus (Gibco BRL). After the introduction of the vector, a drug corresponding to the selection marker of the expression vector was added and the culture was continued. After confirming the colonies, antibody-expressing strains were selected by the method shown in Example 6. Antibody purification from the sorted cells was performed according to Example 8.
  • Example 3 Antigen preparation was performed according to Example 8.
  • an expression plasmid vector of A33 full-length amino acid was prepared.
  • Encoding A33]) NA was prepared by PCR.
  • a) Preparation of full-length A33 expression vector In order to prepare a full-length A33 expression vector, a plasmid vector pAEGFP-N1-GPA33 carrying cDNA encoding A33 was prepared. pAEGFP-N ⁇ GPA33 was prepared by the following method. Full-length A33 DNA (GenBankDNA NMJ05814: SEQ ID NO: 11, protein NP—005305: SEQ ID NO: 12), EcoRI sequence at its 5 'end, polymerase chain for adding Notl sequence and stop codon at its 3' end The reaction (PCR) was performed for modification. Becton Dickinson's Bioscience Hmnan purchased from Clontech
  • Colon Marathon-Ready cDNA is a saddle type, A33-F2 as a primer
  • ⁇ 33 incorporated into EGFP-N1-GPA33 encodes a 977 bp cDNA.
  • gene PCR PCR System 9700 manufactured by Perkin Elma Japan Co., Ltd. was used for reaction temperature control of all PCRs in the Examples.
  • pAEGFP-N1-GPA33 prepared in a) was introduced into two cell lines, FM3A cells (Ministry of Health, Labor and Welfare Research Resource Bank (JCRB) No. 0701) and L929 cells (ATCC No. CCL-1). Two types of A33-expressing cells were prepared.
  • FM3A cells the electro-volition method was used. 20 g of pAEGFP-N ⁇ A33 vector was introduced into 5 ⁇ 10 6 FM3A cells using a BTX electroboretor at 350 V and 950 F and seeded in a 6-well culture plate. . 37 ° (: After incubation for 48 hours under 5.0% carbon dioxide, G418
  • L929 cells were introduced using Trans IT-LT1 (manufactured by Yukara Bio). Gene transfer was performed by the manual method. After culturing at 37 ° C under 5.0% carbon dioxide for 24 hours, G418 (Gibco BRL) was added to lmg / iiiL and cultured for 1 week. As in the case of FM3A cells, the A33 antigen expressed on the cell membrane surface was confirmed using the culture supernatant of AS33 hypridoma. Flow cytometer using AS33 Hypridoma (ATCC No.
  • HB-8779 as primary antibody and R-pliycoerytlirin-labeled goat anti-mouse Ig gamma F (a) 2 antibody (Dakoichi) as secondary antibody FCM (manufactured by Becton Dickinson) was analyzed, and among the cells that had acquired G418 resistance in the transfected cells, cells expressing A33 on the cell membrane surface were selectively sorted.
  • FM3A cells that highly expressed A33 antigen protein were named FM3A / A33, and L929 cells were named L929 / A33.
  • the shA33EX-liFc protein was prepared for use in immunogens or ELI SA for antibody screening.
  • sM33EX-hFc a plasmid vector carrying the cDNA encoding the extracellular region of A33 pTracer-CMV- ImmanFc-A33EXR was made.
  • pTracer-CMV-humanFc-A33EXR was produced by the following method.
  • A33 DNA SEQ ID NO: 11
  • extracellular signal sequence including secretory signal sequence, EcoRI sequence at its 5 'end, and polymerase chain reaction (PCR) to add Notl sequence and stop codon at its 3' end Qualified.
  • PCR polymerase chain reaction
  • CTCGAGCGGCCGCCAGTTCATGGAGGGAGATCTGACG -3 '(SEQ ID NO: 15) was synthesized, and ⁇ -plus
  • PCR reaction of 30 cycles was performed.
  • the synthesized sequence was digested with EcoRI-Notl, isolated as an EcoRI-Notl fragment, and the pTracer-CMV-umanFc vector (modified pTracer-CMV [manufactured by Invitrogen Life Technologies), which had been cleaved with the same enzyme]
  • the plasmid obtained by ligating the FLAG and human IgGl Fc regions into plasmids (I and Apa I sites) was named pTracer-CMV-humanFc-A33EXR.
  • Oligonucleotides such as PCR primers were all synthesized using an automated DNA synthesizer (Model 3948; manufactured by PerkinElmer Co., Ltd., Japan Applied Biosystems) according to the manual [Matteucci, MD and Caruthers, MH (1981) J. Am. Chem. So 103, 3185-3191]. After the synthesis, each oligonucleotide was cleaved from the support and deprotected. The resulting solution was dried and dissolved in distilled water, and stored frozen at ⁇ 20 ° C. until use.
  • the SM33EX-hFc protein expression vector constructed in c) was introduced into a host cell to produce soluble extracellular A33 protein expression cells.
  • Host cells for expression include, for example, dhir deficient strains of CH0 cells (ATCC CRL-9096), CHO-as (KatakuraY., Et al., Cyto technology, 31: 103-109, 1999), HEK293T (ATCC CRL-11268) is used.
  • the vector was introduced into the host cells by electroporation or lipofection.
  • electro-bolition approximately 2 ⁇ g of shA33EX-hFc protein expression vector was linearized with restriction enzymes, and the gene was introduced into 4X10 6 CH0 cells using Bio-Rad electroplioreter under conditions of 350V and 500F. And seeded in a 96-well culture plate. Lipofection was performed according to the manual using LipoiectAMINE Plus (manufactured by Gibco Beerell). After the introduction of Vector 1, a drug corresponding to the selection marker of the expression vector was added, and the culture was continued.
  • the shA33EX-hFc protein was purified from the culture supernatant by the following method. s The culture supernatant containing the A33EX-hFc protein was assayed using Hitrap Protein A FF (Amersham Pharmacia Biotech), PBS as the adsorption buffer, 20 mM sodium citrate, 50 ⁇ sodium chloride ( ⁇ 2 .7) Made. . The elution fraction was adjusted to ppHH55..55 by adding 5500 mmMM solution of sodium natritriphosphate ((ppHH 77 .. 00)). It was. .
  • AA3333 Tatanpung protein solution is prepared using AAmmiiccoonn UUll tt rraa-1155 (manufactured by Amamikoncon Co., Ltd.). Replaced with PPBBSS, and the hole diameter is 00 .. One by one
  • the mouse used for immunization has a homozygous genetic background for both endogenous Ig heavy chain and / c light chain disruption, and a chromosome 14 fragment containing the human Ig heavy chain locus ( SC20) and human Ig / c chain transgene (KCo5) are retained simultaneously.
  • SC20 human Ig heavy chain locus
  • KCo5 human Ig / c chain transgene
  • This mouse was produced by crossing a strain A mouse with a human Ig heavy chain locus with a strain B mouse having a human Ig K chain transgene.
  • Strain A is a mouse strain that is homozygous for both endogenous Ig heavy chain and kappa light chain disruption and carries a descendant transferable chromosome 14 fragment (SC20) .
  • Strain B is a mouse strain (transgenic mouse) that is homozygous for both endogenous Ig heavy chain and / c light chain disruption and carries the human Ig / c chain transgene (KCo5).
  • KCo5 human Ig / c chain transgene
  • A33-expressing FM3A cells (1 X 10 7 cells / mouse) prepared in Example 3 with RIBI adjuvant (manufactured by Corixa) in a human antibody-producing mouse. And immunized for the first time. Since the first immunization, the same cells and RIBI adjuvant were immunized 8 times a week.
  • shA33EX-hFc protein 20 / ⁇ g / mouse was administered to the tail vein and 5 xg / mouse of Recombinant Human IL-6 was subcutaneously administered.
  • SM33EX-hFc protein 10 ig / mouse was mixed with CpG adjuvant (Qiagen) for the first immunization. Since the first immunization, the same protein and CpG adjuvant were immunized twice every two weeks, and then only the same protein was immunized two weeks later. Three days before the spleen acquisition described below, the SM33EX-hFc protein mouse mice were administered via the tail vein.
  • CpG adjuvant Qiagen
  • shA33EX-liFc protein 10_ig / mouse and A33-expressing FM3A cells were immunized intraperitoneally with RIBI adjuvant, and immunized 1 to 4 times every 2 weeks.
  • SM33EX-hFc protein 5 g / mouse was administered intraperitoneally.
  • the spleen was surgically obtained from the immunized mouse and 350 mg / mL sodium bicarbonate,
  • Serum-free DMEM medium (Gipco BRL) containing 50 units / mL penicillin and 50 g / mL streptomycin (hereinafter referred to as “serum-free DMEM medium”) placed in 10 mL, mesh (cell strainer: Falcon) ) Smashed with a spatula above. After centrifuging the cell suspension that passed through the mesh to precipitate the cells, the cells were washed twice with serum-free DMEM medium, then suspended in serum-free EM medium, and the number of cells was measured.
  • Hypridoma is selected from 10% FCS, IL-6 (lOng / mL) (or 10% Hypridoma cloning factor (hereinafter referred to as “HCF”: Biobase)) and hypoxanthine 00, amino This was carried out by culturing in an EM medium containing pterin (A) and thymidine (T) (hereinafter referred to as “HAT”: manufactured by Sigma). In addition, HT (manufactured by Sigma), 10% FCS, 10% FCS,
  • IL-6 (or 10% HCF) -containing orchid EM medium was used to make a single clone by the limiting dilution method.
  • the culture was performed in a 96-well microtiter plate (Becton Dickinson).
  • Screening The selection of the high-pridoma clones that produce anti-A33 rabbit monoclonal antibodies (screening) and the characterization of the human monoclonal antibodies produced by each of the hyperidomas are described in the enzyme-linked immunosorbent assay (ELISA) and flow cytometry (FMC) described below. ).
  • the name of the hybridized clone may represent the name of the antibody.
  • the following high-pridor clones represent single clones: 263A17, 125M10AA, 125M165DAAA, 125M96ABA, 125N26F6AA, 125Q47BA,
  • 125Q54AAAA and 125R5AAAA 1 5M10AA, 125M165DAAA, 125M96ABA, 125N26F6AA, 125Q47BA, 125Q54AAAA and 125R5AAAA are registered in the National Institute of Advanced Industrial Science and Technology Patent Biology Deposit Sen Yuichi (1st East, 1st Street, Tsukuba City, Ibaraki Prefecture, Japan) on August 24, 2004.
  • FERM BP-10107 (Indication for identification: M10), FERM BP-10106 (Indication for identification: M165), Fuji BP-10108 (Indication for identification: M96), FERM BP-10109 (Indication for identification: N26), FERM BP-10104 (Indication for identification: Q47), FERM BP-10105 (Indication for identification: Q54) and FERM ABP-10103 (Indication for identification: R5).
  • Example 6 Selection of clones producing human anti-A33 monoclonal antibody having human immunoglobulin chain (hlg T) and human immunoglobulin light chain / (Ig ⁇ )
  • the plate was washed twice with PBS containing 2% FCS, and TMB chromogenic substrate (manufactured by DAK0) was added to each well, and incubated at room temperature for 20 minutes.
  • 0.5M sulfuric acid 100 xL / well was added to each well to stop the reaction.
  • Absorbance at a wavelength of 450 ⁇ was measured with a microplate reader (1420 ARV0 multilabel counter: manufactured by WALLAC), and antibody-producing clones that showed a positive reaction were selected.
  • FM3A cells not expressing the A33 antigen were used as a negative control. That is, a culture supernatant that reacted with FM3A / A33 cells but did not react with FM3A cells was selected as an antibody-producing clone that showed a positive reaction.
  • the procedure was as follows. Add 50 liters of the shA33EX-Fc protein prepared in Example 3 to lg / ml carbonate buffer pH 9.4, and add to each well of a 96-well microplate for ELISA (Maxison ?, manufactured by Nunk). The shA33EX-hFc protein was adsorbed on the microplate by incubating at room temperature for 1 hour or overnight at 4 ° C. Next, discard the supernatant, add PBS to each well with 10% FCS, and incubate at 37 ° C for 1 hour to bind the sliA33EX-hFc protein. The part which is not done was blocked.
  • microplates were prepared in which each well was coated with SM33EX-hFc protein.
  • Each hybridoma culture supernatant (50 1) was added to each well, allowed to react at room temperature for 1 hour, and each well was washed twice with PBS containing 0.1% Tween20 (PBS-T).
  • PBS-T PBS containing 0.1% Tween20
  • a Hedge anti-human Ig / antibody 50 1 / well, manufactured by The Binding Site) labeled with horseradish radish xydase was added to each well and incubated at 37 ° C for 1 hour.
  • TMB chromogenic substrate solution manufactured by DAK0
  • 0.5M sulfuric acid 100 ⁇ 1 / well
  • Absorbance at a wavelength of 450 nm was measured with a microplate reader (VersaMax, manufactured by Molecular Devices), and antibody-producing clones that showed a positive reaction were selected.
  • FMC hyperidoma culture supernatant to human colon cancer cell line C0LO205 cells expressing A33 antigen was examined.
  • the COLO205 cell line was suspended in Staining Buf fer (SB) in PBS containing 0. ⁇ N 3 and 2 FCS at a concentration of 2 ⁇ lOVml.
  • the cell suspension 50 Zwell was dispensed into 96-wel round bottom plates (Becton Dickinson).
  • the culture supernatant (50 il l) of each hyperidoma was added and incubated for 30 minutes at ice temperature.
  • a negative IgG control antibody was prepared using a human IgGl antibody (manufactured by Sigma) to a concentration of 2 g / ml in a hybridoma culture medium, and incubated at 50 ° C for 30 minutes at ice temperature. .
  • RPE fluorescently labeled goat anti-human IgG F (ab ′) 2 antibody (Southern Biotech) 50 / i 1 was added and incubated at ice temperature for 30 minutes.
  • the suspension was suspended in 300 1 FACS buffer, and the average fluorescence intensity of each cell was measured with FACS (FACScal ibur, Becton Dickinson).
  • FACS Fluorescence intensity of each cell was measured with FACS (FACScal ibur, Becton Dickinson).
  • a microplate was prepared by coating each well with shA33EX-liFc protein. Next, it was washed twice with 0.1% Tween20-containing PBS (PBS-T), and each well was labeled with horseradish peroxidase, respectively.
  • Hedge anti-human IgGl antibody, Hedge anti-human IgG2 antibody Hedge anti-human IgG3 antibody or Hedge anti-human IgG4 antibody (1600, 6400, 25000, 25000-fold dilution, 50 / well, manufactured by The Binding Site, respectively) was added and incubated at room temperature for 1.5 hours.
  • substrate buffer (TMB, lOO ⁇ L / well, DAK0) was added to each well and incubated at room temperature for 20 minutes . Then 0.5M sulfuric acid (100 iL / well) was added to stop the reaction. Absorbance at a wavelength of 450 nm (reference wavelength 570 nm) was measured with a microplate reader (VersaMax, manufactured by Molecular Devices), and the subclass of each clone was determined. Since human anti-A33 antibodies place importance on ADCC and CDC activities, only those with subclass IgG1 were selected.
  • the human anti-A33 monoclonal antibody was purified from the culture supernatant of the hybridoma obtained from Example 6 by the following method.
  • Culture supernatant containing human anti-A33 monoclonal antibody was cultured in SFM medium (Invitrogen) containing 10% ultra low IgGFBS (Invitrogen).
  • Protein A Fast Flow gel (Amasitech) was purified using PBS as an adsorption buffer and 0.02M glycine buffer (PH3.6) as an elution buffer. The elution fraction was adjusted to pH 7.2 by adding lMTris ((8.0).
  • the prepared antibody solution is a Sephadex G25 desalting column (NAP column;
  • human colon cancer cell lines C0L0205, LoVo cells (ATCC No. CCL-229), LS174T cells (ATCC No. CL-188) and NCI-H508 cells (ATCC No. CCL-253) expressing A33 antigen The reactivity of each monoclonal antibody obtained in 8 was examined by FCM.
  • human colon cancer cell line HT-29 cells (ATCC No. HTB-38) not expressing A33 antigen were also used as negative control cells.
  • Each cell line was suspended in Staining Buf fer (SB) in PBS containing 0.1% NaN 3 2% FCS at a concentration of 2 ⁇ 10 6 / ml.
  • FITC fluorescent-labeled goat anti-human IgG F (ab ′) 2 antibody 50 1 manufactured by Southern Biotech was added and incubated at ice temperature for 30 minutes. After washing once with SB, the suspension was suspended in 3001 FACS buffer, and the average fluorescence intensity of each cell was measured with FACS (FACScan, manufactured by Becton Dickinson).
  • Example 8 Whether each monoclonal purified antibody obtained in Example 8 recognizes the same epitope as the mouse anti-A33 antibody was examined in a competition experiment using FCM.
  • the C0L0205 cell line was suspended at a concentration of 2 ⁇ 10 Vml in Staining Buffer (SB) of PBS containing 0.1% NaN 3 and 23 ⁇ 4 FCS. Cell suspension (50 1Z uel) was dispensed into 96-well round bottom plates (Becton Dickinson). Then, add the mouse anti-A33 purified antibody prepared in Example 1 at a concentration of 100 g / ml and the one not added together with 1 ig / ml of each monoclonal purified antibody (50 / l).
  • normal human peripheral blood-derived mononuclear cells were prepared according to a conventional method using Ficoll (Ficoll-PauePLUS: Amersham-Falmacia Biotech). Normal human blood collected in a blood collection bag (manufactured by Terumo) containing sodium citrate solution as an anticoagulant was layered on Ficoll, and mononuclear cells were separated by specific gravity centrifugation (800 G, room temperature for 15 minutes). . The intermediate layer was extracted as mononuclear cells, diluted with PBS, and centrifuged at 200 G for 10 minutes three times to remove platelets remaining in the supernatant.
  • Ficoll Ficoll-PauePLUS: Amersham-Falmacia Biotech.
  • PBMC Normal human peripheral blood mononuclear cells
  • Antibody-mediated cytotoxic activity is NI (having killer activity such as cells or neutrophils).
  • NI having killer activity such as cells or neutrophils.
  • ADCC antibody-dependent cellular cytotoxicity
  • CDC complement-Dependent Cytotoxicity
  • ADCC Atsey uses 500000 healthy human peripheral blood mononuclear cells obtained by the method described in Example 11 for 5,000 51 Cr-labeled target cells in a V-bottom 96-well plate (Course Yuichi). in the whole volume 200 ⁇ L, and 4 hours at 37 ° C, 5% C0 2 presence with each antibody concentration.
  • CDC Atsey uses a 5 % Cr-labeled target cell with 5% human serum complement (Sigma) at a final concentration of 5% in a V-bottom 96-well plate with a total volume of 200 L for each antibody. concentration with 37 ° C, and cultured for 4 hours in the presence 53 ⁇ 4 C0 2.
  • the plate After culturing with both ADCC and CDC assembly, the plate is centrifuged to sink the cells, and each monoclonal antibody is prepared at 0.4-500 ng / ml, and 50 is a 96-well plate containing powder scintillation overnight (L-abdominal aplateTM) -96: Packard) and dried at 55 ° C for 1.5 hours. After confirming drying, the plate was covered with a special cover (TopSealTM-A: 96-well Microplates: Packard), and the dose was measured with a scintillation counter (Topcount: Packard).
  • > 1000 ng / ml, it is indicated as +, and when no specific dissolution rate is observed, it is indicated as 1.
  • ADCC ADCC
  • CA33 and 125Q54AAAA showed high injury activity.
  • 125 M 10AA showed high damage activity.
  • 1st strand cDNA was prepared using 5 S total RNA as a saddle.
  • GSP Gene specific primers
  • reaction solution having the composition described above was subjected to PCR with a final volume of 50 ⁇ 1 with double-distilled water.
  • Amplification of the heavy chain gene of 263A17 was carried out using UPM primer and IgGlp primer (5'-TCTTGTCCACCTTGGTGTTGCTGGGCTTGTG-3 ') (SEQ ID NO: 16) attached to SMART RACE c ⁇ Amplification Kit at 98 ° C for 1 second and 68 ° C for 30 seconds. The cycle was repeated 30 times.
  • amplification of the light chain gene of 263A17 was performed at 98 ° C for 1 second using UPM primer and lik-2 (5'-GTT GAA GCT CTT TGT GAC GGG CGA GC -3 ') (SEQ ID NO: 17) primer.
  • Amplification was performed by repeating 30 cycles of 68 ° C for 30 seconds.
  • reaction conditions were the same as in (2) 1-1.
  • a UPM primer and IgGlp primer SEQ ID NO: 16 were used, and a cycle of 98 seconds at 68 ° C for 30 seconds was repeated 30 times.
  • this reaction solution 1 ⁇ 1 is made into a vertical type, and NUPM primer (SMART RACE cDNA amplification Kit; Becton 'Dickinson Bioscience ⁇ Clontech) and IgG2p / G134 primer (5,-TGC ACG CCG CTG GTC AGG Using GCG CCT GAG TTC C-3 ′) (SEQ ID NO: 18), a cycle of 98 ° C. for 1 second and 68 ° C. for 30 seconds was repeated 20 times.
  • NUPM primer SMART RACE cDNA amplification Kit
  • IgG2p / G134 primer 5,-TGC ACG CCG CTG GTC AGG Using GCG CCT GAG TTC C-3 ′
  • 125M 125, 125M96ABA, 125Q47BA, 125Q54AAAA and 125R5AAAA light chain genes were amplified using UPM primer and hk-2 primer (SEQ ID NO: 17) at a cycle of 98 ° C for 1 second and 68 ° C for 30 seconds. Amplified repeatedly. Furthermore, this reaction solution ll is made into a saddle type, and NUPM primer and 1 ⁇ -5 primer (5'-AGG CAC ACA ACA GAG GCA GTT CCA GAT TTC-3 ') (SEQ ID NO: 19) are used. The cycle of 68 seconds at 68 ° C was repeated 20 times.
  • (2) -3 Amplification of heavy and light chain genes by PCR of Hypridor 125M165DAAA and 125N26F6AA
  • reaction conditions were the same as (2) -1.
  • Amplification of 125M165DAAA and 125N26F6AA heavy chain genes was carried out using UPM primer and hh-2 primer (5′-GCT GGA GGG CAC GGT CAC CAC GCT G-3 ′) (SEQ ID NO: 20), 98 ° C. for 1 second, 68 The cycle of ° C for 30 seconds was repeated 30 times.
  • 1 liter of this reaction solution was made into a saddle type, and NUPM primer and hh-4 primer (5'-GGT GCC AGG GGG AAG ACC GAT GG-3 ') (SEQ ID NO: 21) were used.
  • Second, a cycle of 68 ° C for 30 seconds was repeated 20 times.
  • amplification of 125M165DAAA and 125N26F6AA light chain genes was performed by repeating 30 cycles of 98 ° C for 1 second and 68 ° C for 30 seconds using ⁇ primer and hk-2 primer (SEQ ID NO: 17). . Further, 1 l of this reaction solution was made into a vertical type, and a cycle of 98 ⁇ 0 1 second and 68 ° C 30 seconds was repeated 20 times using NUPM primer and hk-5 primer (SEQ ID NO: 19).
  • PCR fragments of each heavy chain and light chain amplified as described above were collected by ethanol precipitation, recovered by agarose gel electrophoresis, and QIAduick Gel Extraction Kit (DNA purification kit using membrane). (Manufactured by Qiagen).
  • the purified HV amplified fragment or LV amplified fragment was subcloned into the PCR 4 Blunt-T0P0 vector of Zero Blunt T0P0 PC Cloning Kit (Invitrogen), respectively. I prayed for the arrangement.
  • M13FW SEQ ID NO: 3
  • M13RV SEQ ID NO: 4 were used as primers for determining the DNA base sequence.
  • the border between the antibody variable region and the antibody constant region in the heavy chain nucleic acid sequence (SEQ ID NO: 22) is located between the 429th adenine (A) and the 430th guanine (G), and the heavy chain amino acid sequence (SEQ ID NO: 23). ) ⁇ The boundary between the antibody variable region and antibody constant region is located between the 143rd serine (S) and the 144th alanine (A).
  • the boundary between the signal sequence and the antibody variable region in the heavy chain nucleic acid sequence is located between the 57th thymine (T) and 58th guanine (G), and the heavy chain amino acid sequence (SEQ ID NO: 23 ) Is located between the 19th cysteine (C) and the 20th dalamic acid (E).
  • nucleic acid sequence of the variable region of the heavy chain of the 263A17 antibody is from the 58th guanine (G) to the 429th adenine (A).
  • amino acid sequence of the variable region of the heavy chain is from the 20th glutamic acid (E) to the 143rd serine (S).
  • the boundary between the antibody variable region and the antibody constant region in the light chain nucleic acid sequence is located between the 387th adenine (A) and the 388th cytosine (C), and the light chain amino acid sequence (SEQ ID NO: 25).
  • the boundary between the signal sequence and the antibody variable region in the light chain nucleic acid sequence (SEQ ID NO: 24) is located between the 66th cytosine (C) and the 67th guanine (G). 25)
  • the boundary between the signal sequence and the antibody variable region in (22) is the 22nd cysteine (C) and the 23rd Located between sparagic acid (D).
  • the nucleic acid sequence (SEQ ID NO: 24) of the variable region of the 263A17 antibody light chain is from the 67th guanine (G) to the 387th adenine (A).
  • the amino acid sequence of the variable region of the light chain (SEQ ID NO: 25) is from the 23rd aspartic acid (D) to the 129th lysine (K).
  • the DNAs encoding the heavy chain variable region and light chain variable region of 125M10AA, and the amino acid sequences of the heavy chain variable region and light chain variable region are shown below.
  • the boundary between the antibody variable region and the antibody constant region in the heavy chain nucleic acid sequence is located between the 441th adenine (A) and the 442nd guanine (G), and the heavy chain amino acid sequence (SEQ ID NO: 27 ) Is located between the 147th serine (S) and the 148th alanine (A).
  • the boundary between the signal sequence and the antibody variable region in the heavy chain nucleic acid sequence (SEQ ID NO: 26) is the 78th cytosine (C).
  • the nucleic acid sequence (SEQ ID NO: 26) of the variable region of the 125M10AA antibody heavy chain is from the 79th cytosine (C) to the 441th adenine (A).
  • the amino acid sequence of the variable region of the heavy chain (SEQ ID NO: 27) is from the 27th glutamine (Q) to the 147th serine (S).
  • the boundary between the antibody variable region and the antibody constant region in the light chain nucleic acid sequence is located between the 381st adenine (A) and the 382rd cytosine (C), and the light chain amino acid sequence (SEQ ID NO: 29).
  • the boundary between the signal sequence and the antibody variable region in the light chain nucleic acid sequence (SEQ ID NO: 28) is located between the 60th adenine (A) and 61st guanine (G), and the light chain amino acid sequence (SEQ ID NO: In 29), the boundary between the signal sequence and the antibody variable region is located between the 20th dalysin (21) and the 21st dalumate (E).
  • the nucleic acid sequence (SEQ ID NO: 28) of the variable region of the 125M10AA antibody light chain is from the 61st guanine (G) to the 381st adenine (A).
  • the amino acid sequence of the variable region of the light chain (SEQ ID NO: 29) is from the 21st glutamic acid (E) to the 127th lysine (K).
  • the DNAs encoding the 125M165DAAA heavy chain variable region and the light chain variable region, and the amino acid sequences of the heavy chain variable region and the light chain variable region are shown below, respectively.
  • the border between the antibody variable region and the antibody constant region in the heavy chain nucleic acid sequence (SEQ ID NO: 30) is located between the 420th adenine (A) and the 421st guanine (G), and the heavy chain amino acid sequence (SEQ ID NO: 31).
  • the boundary between the signal sequence and the antibody variable region in the heavy chain nucleic acid sequence (SEQ ID NO: 30) is located between the 57th thymine (T) and the 58th cytosine (C), and the heavy chain amino acid sequence (SEQ ID NO: 31).
  • the nucleic acid sequence (SEQ ID NO: 30) of the variable region of the 125M165DAAA antibody heavy chain is from the 58th cytosine (C) to the 420th adenine (A).
  • the amino acid sequence of the variable region of the heavy chain (SEQ ID NO: 31) is from the 20th glutamine (Q) to the 140th serine (S).
  • the nucleic acid sequence (SEQ ID NO: 32) of the variable region of the 125M165DAAA antibody light chain is from the 61st guanine (G) to the 381st adenine (A).
  • the amino acid sequence of the light chain variable region (SEQ ID NO: 33) is from the 21st glutamic acid (E) to the 127th lysine (K). .
  • the boundary between the antibody variable region and the antibody constant region in the heavy chain nucleic acid sequence is located between the 420th adenine (A) and the 421st guanine (G), and the heavy chain amino acid sequence (SEQ ID NO: 35).
  • the nucleic acid sequence (SEQ ID NO: 34) of the variable region of the heavy chain of 125M96ABA antibody is from the 58th cytosine (C) to the 420th adenine (A).
  • the amino acid sequence of the variable region of the heavy chain (SEQ ID NO: 35) is from the 20th glutamine (Q) to the 140th serine (S).
  • nucleic acid sequence (SEQ ID NO: 36) of the variable region of the 125M165DAAA antibody light chain is from the 61st guanine (G) to the 381st adenine (A).
  • amino acid sequence of the light chain variable region (SEQ ID NO: 37) is from the 21st glutamic acid (E) to the 127th lysine (K).
  • the boundary between the antibody variable region and the antibody constant region in the heavy chain nucleic acid sequence (SEQ ID NO: 38) is located between the 420th adenine (A) and the 421st guanine (G), and the heavy chain amino acid sequence (SEQ ID NO: 39).
  • the boundary between the signal sequence and the antibody variable region in the heavy chain nucleic acid sequence (SEQ ID NO: 38) is located between the 57th thymine (T) and the 58th cytosine (C).
  • the boundary between the antibody variable region and the antibody constant region in the light chain nucleic acid sequence is located between the 388th adenine (A) and the 389th cytosine (C), and the light chain amino acid sequence (SEQ ID NO: 41).
  • the boundary between the signal sequence and the antibody variable region in the light chain nucleic acid sequence is located between the 57th thymine (T) and the 58th guanine (G). 41)
  • the boundary between the signal sequence and the antibody variable region is located between the 19th glycine (G) and the 20th dalamic acid (E).
  • the nucleic acid sequence (SEQ ID NO: 40) of the variable region of the 125M96ABA antibody light chain is 58th From guanine (G) to 388th adenine (A).
  • the amino acid sequence of the variable region of the light chain (SEQ ID NO: 41) is from the 20th glutamic acid (E) to the 126th lysine (K).
  • the DNAs encoding the heavy chain variable region and light chain variable region of 125Q47BA, and the amino acid sequences of the heavy chain variable region and the light chain variable region are shown below.
  • CAGCCTGAAG ATTTTGCAAC TTATTACTGC CAACAGTATA ATAGTTACCC GTACACTTTT
  • the border between the antibody variable region and the antibody constant region in the heavy chain nucleic acid sequence (SEQ ID NO: 42) is located between the 420th adenine (A) and the 421st guanine (G), and the heavy chain amino acid sequence (SEQ ID NO: 43).
  • the boundary between the signal sequence and the antibody variable region in the heavy chain nucleic acid sequence (SEQ ID NO: 42) is the 57th thymine (T).
  • the nucleic acid sequence (SEQ ID NO: 42) of the variable region of the 125Q47BA antibody heavy chain is from the 58th guanine (G) to the 420th adenine (A).
  • the amino acid sequence of the variable region of the heavy chain (SEQ ID NO: 43) is from the 20th glutamic acid (E) to the 140th serine (S).
  • the boundary between the antibody variable region and the antibody constant region in the light chain nucleic acid sequence (SEQ ID NO: 44) is located between the 387th adenine (A) and the 388th cytosine (C), and the light chain amino acid sequence (SEQ ID NO: 45).
  • the boundary between the signal sequence and the antibody variable region in the light chain nucleic acid sequence (SEQ ID NO: 44) is located between the 66th thymine (T) and the 67th guanine (G).
  • the boundary between the signal sequence and antibody variable region in No. 45) is located between the 22nd cysteine (C) and the 23rd aspartic acid (D).
  • nucleic acid sequence of the variable region of the 125Q47BA antibody light chain is from the 67th guanine (G) to the 387th adenine (A).
  • amino acid sequence of the variable region of the light chain is from the 23rd aspartic acid (D) to the 129th lysine (K).
  • the DNAs encoding the heavy chain variable region and light chain variable region of 125Q54AAAA and the amino acid sequences of the heavy chain variable region and the light chain variable region are shown below.
  • CAGCCTGAAG ATTTTGCAAC TTATTACTGC CAACAGTATA ATAGTTACCC GTACACTTTT
  • the boundary between the antibody variable region and the antibody constant region in the heavy chain nucleic acid sequence (SEQ ID NO: 46) is located between the 420th adenine (A) and the 421st guanine (G), and the heavy chain amino acid sequence (SEQ ID NO: 47).
  • the boundary between the signal sequence and the antibody variable region in the heavy chain nucleic acid sequence (SEQ ID NO: 46) is located between the 57th thymine (T) and 58th guanine (G), and the heavy chain amino acid sequence (SEQ ID NO: 47).
  • nucleic acid sequence (SEQ ID NO: 46) of the variable region of the 125Q54AAAA antibody heavy chain is from the 58th guanine (G) to the 420th adenine (A).
  • amino acid sequence of the variable region of the heavy chain (SEQ ID NO: 47) is from the 20th glutamic acid (E) to the 140th serine (S).
  • the boundary between the antibody variable region and the antibody constant region in the light chain nucleic acid sequence is located between the 387th adenine (A) and the 388th cytosine (C), and the light chain amino acid sequence (SEQ ID NO: 53).
  • the boundary between the signal sequence and the antibody variable region in the light chain nucleic acid sequence is located between the 66th thymine (T) and the 67th guanine (G).
  • T 66th thymine
  • G 67th guanine
  • the boundary between the signal sequence and the antibody variable region is located between the 22nd cysteine (C) and the 23rd aspartic acid (D).
  • the nucleic acid sequence (SEQ ID NO: 52) of the variable region of the 125MAAAA antibody light chain is from the 67th guanine (G) to the 387th adenine (A).
  • the amino acid sequence of the variable region of the light chain (SEQ ID NO: 53) is from the 23rd aspartic acid (D) to the 129th lysine (K).
  • Example 1 4 Determination of full-length sequence including human antibody heavy chain gene and light chain gene constant region expressed in Hypridor 125N26F6AA and 125M10AA
  • amplification of the light chain ( ⁇ ) of 125N26F6AA is based on the primer for L chain 5 ': N26KA10Minor L Bgl (SEQ ID NO: 64) and primer for L chain 3'-: L_3UTR_823 (5 '-GAAAGATGAGCTGGAGGACCGCAATA-3', SEQ ID NO: 76).
  • the composition of the reaction solution other than the primer was the same as that of Example 13 (2) -1.
  • Each amplified PCR fragment was recovered by ethanol precipitation, then recovered by agarose gel electrophoresis, and purified by QIAQiiick Gel Extension Kit (Qiagen Co., Ltd.).
  • the purified amplified fragments were each subcloned into the PCR 4 Blunt-TOPO vector of Zero Blunt TOPO PCR Cloning Kit (Invitrogen).
  • For the obtained clones use the DNA sequencing reagent, Te Immediately iPM ⁇ DNA Amplification Kit (Amersham Biosciences Co., Ltd.). After preparation, the base sequence of the insert DNA was determined.
  • M13FW (SEQ ID NO: 3), M13RV (SEQ ID NO: 4), 4 (SEQ ID NO: 21), hlil (5'-CCAAGGGCCCATCGGTCTTCCCCCTGGCAC-3 ') (SEQ ID NO: 77) as primers for DNA sequence analysis of human antibody heavy chains ), CMVH903F (5'-GACACCCTCATGATCTCCCGGACC-3 ') (sequence number 78), CMVHR1303 (5'-TGTTCTCCGGCTGCCCATTGCTCT-3') (sequence number 79), hh-6 (5'-GGTCCGGGAGATCATGAGGGTGTCC-3 (SEQ ID NO: 80), hh-2 (SEQ ID NO: 20), H_3UTR1848 (SEQ ID NO: 74), and H-3UTR1875 (SEQ ID NO: 75), and primers for DNA sequence analysis of human antibody light chain ( ⁇ ) M13FW (SEQ ID NO: 3), M13RV (SEQ ID NO: 4
  • CTGGACTCCG ACGGCTCCTT
  • CTTCCTCTAC AGCAAGCTCA CCGTGGACAA GAGCAGGTGG
  • CAGTCTCCAA AGCTCCTCAT CAAGTATGCT TCCCAGTCCT TCTCAGGGGT CCCCTCGAGG
  • CTCATGATCT CCCGGACCCC TGAGGTCACA TGCGTGGTGG TGGACGTGAG CCACGAAGAC
  • VDKKVEPKSC DKTHTCPPCP APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED
  • GAGAGTGTCA CAGAGCAGGA CAGCAAGGAC AGCACCTACA GCCTCAGCAG CACCCTGACG
  • 125M165DAAA and 125N26F6AA antibodies use a plasmid DNA containing the HV chain of the obtained antibody as a saddle and add a restriction enzyme site (5 'terminal SalL 3' unsided Nhel) to the end.
  • the designed primer was used. Specific primers are as follows.
  • the HV of each A33 antibody was amplified by PGR (94C 3 min-94 10 sec, 68 ° C 45 sec (35 cycles) ⁇ n. C 7 min).
  • the amplified DNA fragment was digested with Sall and Nhel and introduced into the N5KG ⁇ Val Lark vector (modified vector of IDEC Pharmaceuticals, N5KG1 (US patent 6001358)) that had been cleaved with the same enzyme.
  • the inserted sequence is subcloned. It was confirmed by sequencing using a vector as a saddle that it was the same as that determined by DNA nucleotide sequence analysis of the processed HV.
  • LV was inserted into the plasmid vector into which the obtained HV was inserted.
  • the obtained plasmid DNA containing the LV chain of the antibody was used as a cage, and a primer designed to add restriction enzyme sites (5'-end BglII, 3'-end BsiWI) for ligation to the ends was used. .
  • Specific primers are as follows.
  • Primer for LV chain 5 ' A33 2-6A2 K L19 Bglll (SEQ ID NO: 60)
  • Primer for LV chain 3 ' A33 2-6A2 K L19 BsiWI (SEQ ID NO: 61)
  • LV of each A33 antibody was amplified by PCR (3 minutes at 94 ⁇ 94 ° C for 10 seconds, 68 ° C for 45 seconds (35 cycles) ⁇ n. C for 7 minutes).
  • the amplified DNA fragment was digested with BglII and BsiWI and introduced into the N5 KG ⁇ HV vector that had been cleaved with the same enzyme.
  • the inserted sequence was confirmed to be identical to that determined by DNA sequencing of the subcloned LV by sequencing the vector as a saddle.
  • plasmid DNA containing the LV chain of the obtained antibody as a saddle and add restriction enzyme sites (5 'terminal BglII, 3' terminal BsiWI) to the ends.
  • restriction enzyme sites 5 'terminal BglII, 3' terminal BsiWI.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Immunology (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Cell Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Microbiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Zoology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Wood Science & Technology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Engineering & Computer Science (AREA)
  • Mycology (AREA)
  • Epidemiology (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne un anticorps ou un fragment de l'anticorps qui peut lier l'antigène A33, en particulier qui attaque une cellule de tumeur exprimant A33 en utilisant un système immune avec ADCC (cytotoxicité cellulaire dépendante des anticorps) ou CDC (cytotoxicité dépendante du complément) et ne produit pas de HAHA ; et un médicament servant à prévenir ou à traiter des tumeurs malignes de différents types jusqu'à présent non soignables, telles que des tumeurs solides, lequel contient l'anticorps ou le fragment d'anticorps tels que décrits ci-dessus. À savoir que l'invention concerne un anticorps capable de lier A33 ou un fragment fonctionnel de celui-ci lequel est un anticorps liant A33 produit par l'hybridome M10 (numéro d'enregistrement : FERM BP-10107), M96 (numéro d'enregistrement : FERM BP-10108), M165 (numéro d'enregistrement : FERM BP-10106), N26 (numéro d'enregistrement : FERM BP-10109), Q47 (numéro d'enregistrement : FERM BP-10104), Q54 (numéro d'enregistrement : FERM BP-10105) ou R5 (numéro d'enregistrement : FERM BP-10107) ou son fragment fonctionnel ; et un médicament servant à prévenir ou à traiter une tumeur lequel médicament contient l'anticorps ci-dessus ou son fragment fonctionnel.
PCT/JP2005/016576 2004-09-06 2005-09-02 Anticorps anti-a33 WO2006028197A1 (fr)

Priority Applications (9)

Application Number Priority Date Filing Date Title
EP05778552A EP1801208A4 (fr) 2004-09-06 2005-09-02 Anticorps anti-a33
CN2005800298522A CN101010427B (zh) 2004-09-06 2005-09-02 抗a33抗体
US11/629,779 US7579187B2 (en) 2004-09-06 2005-09-02 Anti-A33 antibody
AU2005280975A AU2005280975B2 (en) 2004-09-06 2005-09-02 Anti-A33 antibody
CA2579391A CA2579391C (fr) 2004-09-06 2005-09-02 Anticorps anti-a33
JP2006535830A JP4088655B2 (ja) 2004-09-06 2005-09-02 抗ａ３３抗体
HK07111099.0A HK1105993A1 (en) 2004-09-06 2007-10-15 Anti-a33 antibody
US12/053,461 US7432359B2 (en) 2004-09-06 2008-03-21 Anti-A33 antibody
US12/421,431 US20090299039A1 (en) 2004-09-06 2009-04-09 Anti-a33 antibody

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2004259090 2004-09-06
JP2004-259090 2004-09-06

Related Child Applications (3)

Application Number Title Priority Date Filing Date
US11/629,779 A-371-Of-International US7579187B2 (en) 2004-09-06 2005-09-02 Anti-A33 antibody
US12/053,461 Continuation-In-Part US7432359B2 (en) 2004-09-06 2008-03-21 Anti-A33 antibody
US12/421,431 Division US20090299039A1 (en) 2004-09-06 2009-04-09 Anti-a33 antibody

Publications (1)

Publication Number Publication Date
WO2006028197A1 true WO2006028197A1 (fr) 2006-03-16

Family

ID=36036478

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2005/016576 WO2006028197A1 (fr) 2004-09-06 2005-09-02 Anticorps anti-a33

Country Status (10)

Country Link
US (2) US7579187B2 (fr)
EP (2) EP2145954A1 (fr)
JP (2) JP4088655B2 (fr)
KR (1) KR100918746B1 (fr)
CN (1) CN101010427B (fr)
AU (1) AU2005280975B2 (fr)
CA (1) CA2579391C (fr)
HK (1) HK1105993A1 (fr)
TW (1) TW200621803A (fr)
WO (1) WO2006028197A1 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008049098A3 (fr) * 2006-10-19 2009-04-09 Merck & Co Inc Antagonistes d'anticorps du récepteur α1 de l'interleukine-13
US20110182913A1 (en) * 2007-02-14 2011-07-28 Kyowa Hakko Kirin Co., Ltd. Anti fgf23 antibody and a pharmaceutical composition comprising the same
US20120309031A1 (en) * 2007-11-07 2012-12-06 Celldex Therapeutics Inc. Antibodies that bind human dendritic and epithelial cell 205 (dec-205)
JP2016504370A (ja) * 2012-12-28 2016-02-12 プレシジョン・バイオロジクス・インコーポレイテッド 結腸癌及び膵臓癌の診断ならびに治療のための、ヒト化モノクローナル抗体及び使用方法
US9550824B2 (en) 2008-07-08 2017-01-24 Geneuro Sa Therapeutic use of specific ligand in MSRV associated diseases

Families Citing this family (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2207808B1 (fr) 2007-11-02 2013-05-08 Novartis AG Molécules améliorées de liaison à nogo-a et utilisation pharmaceutique de celles-ci
WO2014074528A2 (fr) * 2012-11-06 2014-05-15 Medimmune, Llc Polythérapies à l'aide de molécules de liaison anti-pseudomonas psl et pcrv
EP2777714A1 (fr) 2013-03-15 2014-09-17 NBE-Therapeutics LLC Procédé de production d'un conjugué de ligand immunologique/charge utile à l'aide d'une enzyme transpeptidase spécifique pour une séquence
US11746159B2 (en) * 2015-02-10 2023-09-05 Minerva Biotechnologies Corporation Humanized anti-MUC1* antibodies
EP3684369A4 (fr) * 2017-09-23 2021-06-23 Memorial Sloan Kettering Cancer Center Compositions d'anticorps a33 et leurs méthodes d'utilisation en radioimmunothérapie
CN110540591A (zh) * 2019-08-09 2019-12-06 无锡傲锐东源生物科技有限公司 一种抗糖蛋白A33（Glycoprotein A33）单克隆抗体及其免疫检测应用
AU2021381768A1 (en) * 2020-11-18 2023-06-22 Memorial Sloan Kettering Cancer Center Anti-gpa33 multi-specific antibodies and uses thereof

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS62111697A (ja) * 1985-04-19 1987-05-22 スロ−ン　−　ケツタリング　インステイテユ−ト　フオ−　キヤンサ−　リサ−チ ヒトの消化器ガンに対するモノクロ−ナル抗体
JP2004515230A (ja) * 2000-11-30 2004-05-27 メダレックス，インコーポレーテッド ヒト抗体作製用トランスジェニック染色体導入齧歯動物

Family Cites Families (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB8308235D0 (en) 1983-03-25 1983-05-05 Celltech Ltd Polypeptides
US4816567A (en) 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations
US5851526A (en) 1985-04-19 1998-12-22 Ludwig Institute For Cancer Research Methods of treating colon cancer utilizing tumor-specific antibodies
US5160723A (en) 1985-04-19 1992-11-03 Sloan-Kettering Institute For Cancer Research Method of imaging colorectal carcinoma lesion and composition for use therein
US5431897A (en) 1985-04-19 1995-07-11 Sloan-Kettering Institute For Cancer Research Method of imaging colorectal carcinoma lesion and composition for use therein
GB8607679D0 (en) 1986-03-27 1986-04-30 Winter G P Recombinant dna product
FI884924L (fi) 1987-10-28 1989-04-29 Oncogen Humanimmuglobulin som producerats med hybrid-dna-teknik.
US5530101A (en) 1988-12-28 1996-06-25 Protein Design Labs, Inc. Humanized immunoglobulins
GB2278357B (en) * 1992-12-10 1997-03-05 Celltech Ltd Humanised antibodies directed against A33 antigen
US6307026B1 (en) * 1992-12-10 2001-10-23 Celltech Limited Humanized antibodies directed against A33 antigen
US6001358A (en) 1995-11-07 1999-12-14 Idec Pharmaceuticals Corporation Humanized antibodies to human gp39, compositions containing thereof
AU1102401A (en) 1999-10-22 2001-05-08 Ludwig Institute For Cancer Research Methods for reducing the effects of cancers that express a33 antigen using a33 antigen specific immunoglobulin products
US6643550B2 (en) * 2000-12-15 2003-11-04 Cardiac Pacemakers, Inc. Multi-polar connector
JP2004259090A (ja) 2003-02-27 2004-09-16 Toshiba Corp 情報処理装置および情報処理方法
US7432359B2 (en) * 2004-09-06 2008-10-07 Kirin Pharma Kabushiki Kaisha Anti-A33 antibody
JP4976741B2 (ja) * 2006-05-16 2012-07-18 株式会社東芝 平面アンテナ

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS62111697A (ja) * 1985-04-19 1987-05-22 スロ−ン　−　ケツタリング　インステイテユ−ト　フオ−　キヤンサ−　リサ−チ ヒトの消化器ガンに対するモノクロ−ナル抗体
JP2004515230A (ja) * 2000-11-30 2004-05-27 メダレックス，インコーポレーテッド ヒト抗体作製用トランスジェニック染色体導入齧歯動物

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
See also references of EP1801208A4 *
TOMIZUKA K. ET AL: "Double trans-chromosomic mice: maintenance of two individual human chromosome fragments containing Ig heavy and kappa loci and expression of fully human antibodies", PROC.NATL.ACAD.SCI. USA, vol. 97, no. 2, 18 January 2000 (2000-01-18), pages 722 - 727, XP002150871 *
WELT S. ET AL: "Preliminary report of a phase I study of combination chemotherapy and humanized A33 antibody immunotherapy in patients with advanced colorectal cancer", CLIN. CANCER RES., vol. 9, no. 4, April 2003 (2003-04-01), pages 1347 - 1353, XP002995035 *

Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8207304B2 (en) 2006-10-19 2012-06-26 Csl Limited Antibody antagonists of interleukin-13 receptor α1
WO2008049098A3 (fr) * 2006-10-19 2009-04-09 Merck & Co Inc Antagonistes d'anticorps du récepteur α1 de l'interleukine-13
US9290569B2 (en) * 2007-02-14 2016-03-22 Kyowa Hakko Kirin Co., Ltd. Anti FGF23 antibody and a pharmaceutical composition comprising the same
US20110182913A1 (en) * 2007-02-14 2011-07-28 Kyowa Hakko Kirin Co., Ltd. Anti fgf23 antibody and a pharmaceutical composition comprising the same
US10202446B2 (en) 2007-02-14 2019-02-12 Kyowa Hakko Kirin Co., Ltd. Anti FGF23 antibody and a pharmaceutical composition comprising the same
US8586720B2 (en) * 2007-11-07 2013-11-19 Celldex Therapeutics Inc. Antibodies that bind human dendritic and epithelial cell 205 (DEC-205)
US9624300B2 (en) 2007-11-07 2017-04-18 Celldex Therapeutics Inc. Antibodies that bind human dendritic and epithelial cell 205 (DEC-205)
US20120309031A1 (en) * 2007-11-07 2012-12-06 Celldex Therapeutics Inc. Antibodies that bind human dendritic and epithelial cell 205 (dec-205)
US9550824B2 (en) 2008-07-08 2017-01-24 Geneuro Sa Therapeutic use of specific ligand in MSRV associated diseases
US9815888B2 (en) 2008-07-08 2017-11-14 Geneuro Sa Therapeutic use of specific ligand in MSRV associated diseases
US10059758B2 (en) 2008-07-08 2018-08-28 Geneuro Sa Therapeutic use of specific ligand in MSRV associated diseases
JP2016504370A (ja) * 2012-12-28 2016-02-12 プレシジョン・バイオロジクス・インコーポレイテッド 結腸癌及び膵臓癌の診断ならびに治療のための、ヒト化モノクローナル抗体及び使用方法
US9938344B2 (en) 2012-12-28 2018-04-10 Precision Biologics, Inc. Humanized monoclonal antibodies and methods of use for the diagnosis and treatment of colon and pancreas cancer
JP2019080572A (ja) * 2012-12-28 2019-05-30 プレシジョン・バイオロジクス・インコーポレイテッド 結腸癌及び膵臓癌の診断ならびに治療のための、ヒト化モノクローナル抗体及び使用方法
US10689443B2 (en) 2012-12-28 2020-06-23 Precision Biologics, Inc. Humanized monoclonal antibodies and methods of use for the diagnosis and treatment of colon and pancreas cancer
JP7003071B2 (ja) 2012-12-28 2022-02-04 プレシジョン・バイオロジクス・インコーポレイテッド 結腸癌及び膵臓癌の診断ならびに治療のための、ヒト化モノクローナル抗体及び使用方法
US11505604B2 (en) 2012-12-28 2022-11-22 Precision Biologics, Inc. Humanized monoclonal antibodies and methods of use for the diagnosis and treatment of colon and pancreas cancer

Also Published As

Publication number Publication date
CA2579391C (fr) 2010-10-26
JPWO2006028197A1 (ja) 2008-05-08
TWI359154B (fr) 2012-03-01
CA2579391A1 (fr) 2006-03-16
JP4088655B2 (ja) 2008-05-21
US20070141054A1 (en) 2007-06-21
US20090299039A1 (en) 2009-12-03
EP1801208A4 (fr) 2009-04-01
US7579187B2 (en) 2009-08-25
TW200621803A (en) 2006-07-01
AU2005280975A1 (en) 2006-03-16
KR100918746B1 (ko) 2009-09-24
CN101010427A (zh) 2007-08-01
EP1801208A1 (fr) 2007-06-27
KR20070088570A (ko) 2007-08-29
CN101010427B (zh) 2011-07-27
AU2005280975B2 (en) 2009-06-18
HK1105993A1 (en) 2008-02-29
JP2008138004A (ja) 2008-06-19
EP2145954A1 (fr) 2010-01-20

Similar Documents

Publication Publication Date Title
AU2021200264B2 (en) Anti-pd-1 antibodies
US20090299039A1 (en) Anti-a33 antibody
KR101751965B1 (ko) 암 치료를 위한 claudin-18에 대한 모노클로날 항체
AU2003230183B2 (en) Humanized immunomodulatory monoclonal antibodies for the treatment of neoplastic disease or immunodeficiency
CA2648618C (fr) Nouvel anticorps anti-cd98
WO2012147713A1 (fr) Anticorps anti-b7-h3
CN110964108A (zh) 抗pd-l1抗体
CN101312989A (zh) 用于治疗癌症的针对密蛋白-18的单克隆抗体
CN101124240A (zh) 肝细胞生长因子的特异性结合物
KR20220071177A (ko) 인간화 항-vegf 단클론 항체
US7432359B2 (en) Anti-A33 antibody
KR20220131279A (ko) 항lag3단일 클론 항체 및 그 제조 방법과 응용
AU2020316495A1 (en) Humanized anti-VEGF Fab antibody fragment and use thereof
CN113226371A (zh) 与TfR结合的双特异性抗体
CN117425500A (zh) 抗dll3抗体-药物缀合物
RU2802960C2 (ru) Fab ФРАГМЕНТ ГУМАНИЗИРОВАННОГО АНТИТЕЛА ПРОТИВ VEGF И ЕГО ПРИМЕНЕНИЕ
CN115028722B (zh) 抗tslp抗体及其制备方法和用途
EP4541816A1 (fr) Anticorps anti-cldn18.2, composition pharmaceutique et utilisation associées

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KM KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NA NG NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SM SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): BW GH GM KE LS MW MZ NA SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LT LU LV MC NL PL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

WWE Wipo information: entry into national phase

Ref document number: 2006535830

Country of ref document: JP

121 Ep: the epo has been informed by wipo that ep was designated in this application
WWE Wipo information: entry into national phase

Ref document number: 2007141054

Country of ref document: US

Ref document number: 11629779

Country of ref document: US

WWE Wipo information: entry into national phase

Ref document number: 200580029852.2

Country of ref document: CN

Ref document number: 2579391

Country of ref document: CA

NENP Non-entry into the national phase

Ref country code: DE

WWE Wipo information: entry into national phase

Ref document number: 2005778552

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 2005280975

Country of ref document: AU

WWE Wipo information: entry into national phase

Ref document number: 1020077007788

Country of ref document: KR

WWP Wipo information: published in national office

Ref document number: 11629779

Country of ref document: US

WWP Wipo information: published in national office

Ref document number: 2005778552

Country of ref document: EP

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载